Conservation of Structure and Function in Vertebrate C-FLIP Proteins Despite Rapid Evolutionary Change

Biochemistry and Biophysics Reports 3 (2015) 175–189

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Biochemistry and Biophysics Reports

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Conservation of structure and function in vertebrate c-FLIP proteins despite rapid evolutionary change

Kazuhiro Sakamaki a,n, Naoyuki Iwabe b, Hiroaki Iwata c,1, Kenichiro Imai d, Chiyo Takagi e, Kumiko Chiba a, Chisa Shukunami f,2, Kentaro Tomii d, Naoto Ueno e a Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan b Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan c Multi-scale Research Center for Medical Science, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan d Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064, Japan e Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan f Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan article info abstract

Article history: Cellular FLICE-like inhibitory protein (c-FLIP, gene symbol CFLAR)wasfirst identified as a negative reg- Received 10 July 2015 ulator of death receptor-mediated apoptosis in mammals. To understand the ubiquity and diversity of the Received in revised form c-FLIP protein subfamily during evolution, c-FLIP orthologs were identified from a comprehensive range 5 August 2015 of vertebrates, including birds, amphibians, and fish, and were characterized by combining experimental Accepted 5 August 2015 and computational analysis. Predictions of three-dimensional protein structures and molecular phylo- Available online 7 August 2015 genetic analysis indicated that the conserved structural features of c-FLIP proteins are all derived from an Keywords: ancestral caspase-8, although they rapidly diverged from the subfamily consisting of caspases-8, -10, and Apoptosis -18. The functional role of the c-FLIP subfamily members is nearly ubiquitous throughout vertebrates. Caspase-8 Exogenous expression of non-mammalian c-FLIP proteins in cultured mammalian cells suppressed death Embryogenesis receptor-mediated apoptosis, implying that all of these proteins possess anti-apoptotic activity. Fur- Evolution κ κ thermore, non-mammalian c-FLIP proteins induced NF- B activation much like their mammalian NF- B fi Pseudocatalytic triad counterparts. The CFLAR mRNAs were synthesized during frog and sh embryogenesis. Overexpression of a truncated mutant of c-FLIP in the Xenopus laevis embryos by mRNA microinjection caused thorax edema and abnormal constriction of the abdomen. Depletion of cflar transcripts in zebrafish resulted in developmental abnormalities accompanied by edema and irregular red blood cell flow. Thus, our results demonstrate that c-FLIP/CFLAR is conserved in both protein structure and function in several vertebrate species, and suggest a significant role of c-FLIP in embryonic development. & 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Apoptotic cell death is essential for multicellular organisms. In the apoptosis process, a family of cysteine proteases known as Abbreviations: CARD, caspase-recruitment domain; CASc, Caspase, interleukin-1 caspases are activated and work as executors to induce the pro- β converting enzyme homologs; c-FLIP, cellular FLICE-like inhibitory protein; CHX, cycloheximide; DED, death effector domain; EGFP, enhanced green fluorescent teolytic cleavage of many critical proteins leading cells to suicide protein; FADD, Fas-associated death domain protein; MO, morpholino oligonu- [1]. The activation of caspases converges from two major signaling cleotide; NF-κB, Nuclear factor-kappa B; ODC, ornithine decarboxylase; PCR, pathways: the extrinsic signaling pathway initiated by ligation of polymerase chain reaction; TRAF2, tumor necrosis factor receptor-associated factor the cell surface death receptors such as Fas (also called APO-1/ 2; tubα6, tubulin α6; RT-PCR, reverse transcription-polymerase chain reaction n Corresponding autor. Fax: þ81 75 753 9235. CD95) and the intrinsic signaling pathway triggered by cyto- E-mail address: [email protected] (K. Sakamaki). chrome c release from mitochondria into the cytosol by apoptotic 1 Present address: Department of Molecular Life Science, Institute of Biome- stimuli [2,3]. The Fas-mediated pathway is well characterized as dical Research and Innovation (IBRI), Foundation for Biomedical Research and In- the representative extrinsic pathway. Oligomerization of Fas by its novation (FBRI), Kobe 650-0047, Japan. natural ligand or an agonistic antibody recruits an adapter mole- 2 Present address: Department of Molecular Biology and Biochemistry, Division of Basic Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, cule, Fas-associated death domain protein (FADD) to the death Hiroshima 734-8553, Japan. domain within the intracellular region. The initiator caspase, http://dx.doi.org/10.1016/j.bbrep.2015.08.005 2405-5808/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 176 K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189 caspase-8 (CASP8), which contains tandem death effector domain 2.2. DNA sequencing (DED) motifs and a protease domain, caspase, interleukin-1 β converting enzyme homologs (CASc), in turn associates with FADD To search for non-mammalian orthologs of the human CFLAR by way of an interaction between their respective DEDs. In the gene in the NCBI DNA database, the TBLASTN program [25] was complex composed of Fas-FADD-CASP8, CASP8 progresses through used, and four candidate EST clones from chicken, African clawed an auto-processing step and transforms to an active form. Acti- frog, medaka, and stickleback were identified and obtained from vated CASP8 subsequently processes and activates downstream distributors as described in our previous study [26]. The nucleo- molecules, resulting in cell death. tide sequence of these clones was confirmed on both strands using Cellular FLICE-like inhibitory protein (c-FLIP; also called CFLAR, TaqDyeDeoxyterminator Cycle sequencing (Applied Biosystems CLARP, FLAME-1, I-FLICE, Casper, CASH, and Usurpin, gene symbol Inc., Foster City, CA) on automated DNA sequencers (3130XL Ge- – CFLAR) is a negative regulator of Fas-induced apoptosis [4 11]. netic Analyzer, Applied Biosystems Inc.). Mammalian c-FLIP exists as two alternatively spliced isoforms: c-FLIP(L) (long form of c-FLIP) is homologous to CASP8, except it lacks critical amino acids for proteolytic activity [8]; c-FLIP(S) 2.3. Database search (short form of c-FLIP) consists of only two DED motifs. In humans, a third isoform, c-FLIP Raji (c-FLIP(R)) was also identified [12]. The following web sites were used to access nucleotide and These isoforms are able to bind directly to CASP8 by homophilic protein sequence databases: NCBI (http://blast.ncbi.nlm.nih.gov/ interactions through their DED domains, and thereby regulate the Blast.cgi), Ensembl (http://www.ensembl.org/index.html), Xen- activation of CASP8 and apoptosis [12,13]. c-FLIP can also induce base (http://www.xenbase.org/entry/), and ZFIN (http://zfin.org/ Nuclear Factor-kappa B (NF-κB) activation [14,15]. In previous action/blast/blast). Proteins related to known c-FLIP and its para- studies, we and other groups have reported that both CFLAR and logous proteins were identified using TBLASTN [27]. Domain CASP8 genes, along with their homologous genes caspase-10 searches were performed using either Simple Modular Archi- (CASP10) and caspase-18 (CASP18), localize to the same chromo- tecture Research ToolSMART (SMART) (http://smart.embl-heidel somal region in marsupials, birds, reptiles, and amphibians [16– berg.de/smart/set_mode.cgi?NORMAL=1) or the Pfam database 19]. Therefore, it is likely that these four genes are derived from an (http://pfam.sanger.ac.uk/search). Multiple sequence alignments ancestral gene by duplication during evolution. Interestingly, the were performed using Clustal W (http://www.genome.jp/tools/ CASP18 gene is deleted in eutherian mammals, and the Casp10 clustalw/). gene is further deleted in rodents [16–18]. However, the CFLAR gene as well as the CASP8 gene is found from fish to mammals, 2.4. Molecular phylogenetic analysis suggesting that both are requisite in vertebrates. In this study, we show both the conservation of the three-di- To construct a molecular phylogenetic tree, we collected pub- mensional structure of c-FLIP proteins in vertebrates and the lished or predicted amino acid sequences of c-FLIP and its para- functional universality in regulation of apoptosis and NF-κB signaling. Furthermore, we clarify the action of c-FLIP proteins in logs, CASP8, CASP10, CASP18, and CARD (caspase-recruitment embryos of non-mammalian vertebrates, pointing to an important domain)-casp8 from several databases for mammals [human role during embryonic development. Based on these results, we (Homo sapiens), mouse (Mus musculus), opossum (Monodelphis propose the uniqueness of c-FLIP relative to its paralogous mole- domestica), pig (Sus scrofa), and rat (Rattus norvegicus)], birds cules, as well as its conserved function through vertebrate [chicken (Gallus gallus) and turkey (Meleagris gallopavo)], reptiles evolution. [anole lizard (Anolis carolinensis) and alligator (Alligator mississippiensis)], amphibian [African clawed frog (X. laevis)], fish [catshark (Scyliorhinus canicula), coelacanth (Latimeria chalumnae), 2. Materials and methods medaka (Oryzias latipes), skate (Leucoraja erinacea), spotted gar (Lepisosteus oculatus), stickleback (Gasterosteus aculeatus), zebra- 2.1. Animals, cell lines, and reagents fish (D. rerio), and lamprey (Petromyzon marinus)], and chordates [ascidian (Ciona intestinalis) and lancelet (Branchiostoma floridae)] Adult wild-type Xenopus laevis were purchased from Hama- (Table A1). For phylogenetic reconstruction, the amino acid se- matsu Seibutsukyozai Co. (Shizuoka, Japan). In vitro fertilization of quences were multiply aligned using MAFFT [28] and manually X. laevis eggs was performed as previously described [20]. Ferti- inspected. Based on the unambiguously aligned amino acid posi- lized embryos were dejellied in 3% cysteine hydrochloride, washed tions in the protease-like/protease (CASc*/CASc) domain, the several times in water and used for several experiments. Devel- phylogenetic tree was constructed by Randomized Axelerated oping embryo stages were determined according to the scheme Maximum Likelihood (RAxML) [29] using the GAMMA-WAG ami- fi described by Nieuwkoop and Faber [21]. Wild-type zebra sh Da- no acid substitution model. nio rerio AB and RW lines were kept in a light and temperature controlled facility and maintained at optimal breeding conditions [22]. Embryos prepared by breeding were staged according to 2.5. Comparative modeling of non-mammalian c-FLIP proteins and Kimmel et al. [23]. Human cervical carcinoma HeLa cells and comparison with human c-FLIP embryonal kidney HEK293 cells were cultured in Dulbecco’s Modified Eagle’s medium with 10% fetal calf serum. An agonistic To build structural models of a CASc* domain of non-mam- anti-human Fas antibody, CH-11, was prepared as previously de- malian c-FLIP proteins, we used the crystal structure of human scribed [24]. Anti-Flag (M2, Sigma-Aldrich, St. Louis, MO), anti-HA c-FLIP, 3H13 [30], which has more than 25% sequence identity (HA124, Nacalai Tesque, Kyoto, Japan), anti-Myc (9E10, Roche Di- with other c-FLIPs, as a template. The models were built using the agnostics GmbH, Mannheim, Germany), anti-actin (MAB1501R, Molecular Operating Environment (MOE) software package (MOE Chemicon International Inc., Temecula, CA), and HRP-conjugated version 2012.10, Chemical Computing Group Inc., Tokyo), based on anti-mouse IgG (Cell Signaling Technology, Danvers, MA, USA) the alignments calculated by FORTE, a profile–profile comparison antibodies and anti-FLAG M2 affinity gel (Sigma-Aldrich) were method for protein structure prediction [31], and were super- purchased. imposed onto the structure of human c-FLIP. K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189 177

2.6. Construction and transfection of plasmids transfection, cells were stimulated with 250 ng/ml of CH11 and 5 μg/ml of cycloheximide (CHX). After an additional 8 h incuba- To express chicken, African clawed frog, zebrafish, and medaka tion, cells were fixed in PBS containing 3.7% formaldehyde, washed c-FLIP proteins in mammalian cell lines, plasmid constructs with PBS, and observed by phase-contrast and fluorescence mi- pME18S-Flag/GgFLIP, pME18S-Flag/XlFLIP, pME18S-Flag/DrFLIP, croscopy (DMIRE2, Leica Microsystems, Wetzlar, Germany). and pME18S-Flag/OlFLIP were generated by fusion of the respective CFLAR polymerase chain reaction (PCR)-amplified coding 2.9. Reporter assays for detection of NF-κB activation sequences from EST clones of these species and the Flag-tag sequence, followed by insertion into a mammalian expression vector, The ability of non-mammalian c-FLIP proteins to activate NF-κB pME18S [32]. The pCMV-Flag/HsFLIP plasmid was generated by in- was examined by a Dual-Luciferase Reporter Assay System (Pro- frame inserting human CFLAR cDNA into pCMV-Tag2B (Agilent mega, Madison, WI). Briefly, the plasmids carrying the non- Technologies, Santa Clara, CA). The pExpress1-GaFLIP plasmid, mammalian CFLAR cDNAs were transfected with either reporter which carries full-length stickleback cflar cDNA, was obtained plasmid pTAL-Luc or pNFκB-Luc in conjunction with another re- from Thermo Fisher Scientific Open Biosystems (Huntsville, AL) porter plasmid pRL-TK into HEK293 cells. After 48 h cultivation, and used for the expression of stickleback c-FLIP. The pCS2-XlFLIP cells were lysed in lysis buffer and the cell lysates were assayed for (DEDs) plasmid was generated by inserting a DNA fragment am- the activities of firefly and sea pansy luciferases, using a multilabel plified by PCR into an expression vector, pCS2. The pME18S-HA/ counter (ARVO-SX, PerkinElmer Life Science-Wallac Oy, Turku, hFADD plasmid was generated by inserting the human FADD Finland). coding sequence fused with an HA-tag sequence into pME18S, and The NF-κB activation by non-mammalian c-FLIP proteins was pME18S-Myc/TRAF2 was generated by inserting the mouse Traf2 also examined with another reporter plasmid, pCMV-EGFP/p65 coding sequence fused with a Myc-tag sequence. The pCAG-Venus [35]. By co-transfecting pCMV-EGFP/p65 with plasmids carrying plasmid, which was used to distinguish between transfected and the non-mammalian CFLAR cDNAs, the cellular localization of an untransfected cells, and the pCAG-p35 plasmid, which was used to EGFP/p65 protein was evaluated by microscopy after 48 h inhibit caspase activation in transfected cells, have been previously cultivation. described [33,34]. Transfection of plasmid DNAs into HeLa and HEK293 cells was performed using Lipofectamine and PLUS Re- 2.10. Reverse transcription-polymerase chain reaction (RT-PCR) agent or LTX Reagent (Invitrogen, Carlsbad, CA), according to the analyses manufacturer’s instructions. Total RNAs were isolated from X. laevis embryos collected at 2.7. Immunoblotting and immunoprecipitation analyses various stages and RT-PCR was performed as described previously [36]. Briefly, first-strand cDNAs were amplified with two sets of To detect human, chicken, African clawed frog, and zebrafish primers: primers (forward: 5’-GCCGAGCGATATGTGGTTAGAA-3’, c-FLIP proteins in mammalian culture cells, HeLa cells were reverse: 5’- GCTTCAACTGGGATTTTAGGCG-3’) for the detection of transfected with pCMV-Flag/HsFLIP, pME18S-Flag/GgFLIP, CFLAR transcripts, primers (forward: 5’-CAGCTAGCTGTGGTGTGG- pME18S-Flag/XlFLIP, pME18S-Flag/DrFLIP, or pME18S, cultured for 3’ and reverse: 5’-CAACATGGAAACTCACACC-3’) for the detection 48 h, harvested, and suspended in lysis buffer containing 50 mM of ornithine decarboxylase (ODC) transcripts as an internal control.

Tris–HCl (pH 7.5), 20 mM MgCl2, 0.5% Nonidet P-40, 150 mM NaCl, PCR consisted of an initial denaturation at 94 °C for 3 min followed and the protease inhibitor cocktails (Nacalai Tesque). After re- by 20 (CFLAR)or18(ODC) cycles of 94 °C for 1 min, 57 °C for 1 min, moving cell debris by centrifugation, the cell lysates were added to 72 °C for 1 min, and a final extension at 72 °C for 3 min. Amplified Laemmli sample buffer, denatured by boiling, resolved by SDS- PCR products were analyzed by 7.5 % acrylamide-gel PAGE, and probed by immunoblotting with anti-Flag and anti-ac- electrophoresis. tin antibodies, respectively. After incubation with a secondary Total RNAs were also isolated from zebrafish embryos collected HRP-conjugated anti-mouse IgG antibody, immune complexes at various stages as described previously [16]. For RT-PCR analysis, were visualized with ImmobilonTM Western chemiluminescent 2 μg of each RNA sample was used for first-strand cDNA synthesis reagent (Millipore Corporation, Billerica, MA), using a luminescent with an oligo-dT primer on Ready-To-Go RT-PCR beads (Amer- image analyzer (LAS-3000, Fujifilm, Tokyo, Japan). sham Biosciences, Arlington Heights, IL), according to the manu- To detect the physical association of non-mammalian c-FLIP facturer's recommendations. First-strand cDNAs were amplified proteins with human FADD or mouse TRAF2 in cells, HEK293 cells with two sets of primers: primers (forward: 5’-GAAGCGCTAT- were transfected with plasmids encoding non-mammalian Flag/c- GATTTACTACGC-3’, reverse: 5’-TTTTGGACACTTGGTTGCGTT-3’) for FLIP and either plasmid pME18S-HA/hFADD or pME18S-Myc/ the detection of cflar transcripts, and primers (forward: 5’- TRAF2. In the case of transfection with pME18S-HA/hFADD, the CTGTTGACTACGGAAAGAAGT-3’, reverse: 5'-TATGTGGACGCTC- plasmid pCAG-p35 was co-transfected to prevent cell death of TATGTCTA-3') for tubulin α6 (tubα6) transcripts as an internal FADD-expressing cells. Two days after transfection, cell lysates control. PCR consisted of an initial denaturation at 96 °C for 3 min were prepared from transfected cells, incubated with anti-FLAG followed by 35 cycles of 94 °C for 45 s, 58 °C for 60 s, 72 °C for 90 s, M2 affinity gel (Sigma-Aldrich) at 4 °C overnight, and the beads and a final extension at 72 °C for 5 min. were washed 5 times with lysis buffer. The immunoprecipitated proteins were eluted with Laemmli sample buffer and analyzed by 2.11. Microinjection of synthetic mRNA into Xenopus embryos SDS-PAGE and immunoblotting with the antibodies indicated in the figure legends. To express a truncated form of c-FLIP in X. laevis embryos, capped mRNA was synthesized using a template DNA, pCS2-XlFLIP 2.8. Cytotoxicity assays (DEDs), with the mMESSAGE mMACHINE Kit (Ambion, Austin, TX), and purified by passing through a Sephadex G-50 column (GE For cytotoxicity assays, HeLa cells were transiently transfected Healthcare, Arlington Heights, IL) as described previously [37]. with pCMV-Flag/HsFLIP, pME18S-Flag/GgFLIP, pME18S-Flag/XlFLIP, Microinjection of synthetic mRNA into embryos was performed at pME18S-Flag/DrFLIP, pME18S-Flag/OlFLIP, pExpress1-GaFLIP, or the four-cell stage. The injected embryos were statically cultured pME18S in conjunction with pCAG-Venus. At 48 h after at 17 °C overnight in 3% Ficoll and 0.1 Steinberg’s solution 178 K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189

[460 nM Tris–HCl (pH 7.2), 5.8 mM NaCl, 67 nM KCl, 34 nM lost during the evolution of various mammalian lineages [18,19].

Ca(NO3)2, 83 nM MgSO4, and 0.1 mg/ml Kanamycin], then trans- These observations suggest that the CFLAR genes might have di- ferred in 0.1 Marc’s Modified Ringers [0.5 mM HEPES (pH 7.8), 10 verged during evolution. To define the genomic structure of the mM NaCl, 0.2 mM KCl, 0.2 mM CaCl2, 0.1 mM MgSO4, and 0.1 mM CFLAR genes encoding c-FLIP proteins in vertebrates, we examined EDTA] and incubated at 23 °C until the proper stages for the genomic sequences published in the NCBI, Ensembl, and experiments. Xenbase genomic databases, and identified genomic sequences, which cover the CFLAR cDNA sequence, from chicken, African 2.12. Microinjection of morpholino oligonucleotide into zebrafish clawed frog, medaka, stickleback, and zebrafish (Table A2). Com- embryos parisons of these genomic and corresponding cDNA sequences clarified the similar organization of the CFLAR genes in these five The antisense morpholino oligonucleotide (MO) for the coding animals. The CFLAR genes consist of nine to eleven exons. In every sequence containing the start ATG codon of the zebrafish cflar case, however, the CFLAR coding region basically covered nine gene (5’-CCAGAGTAGAAAATCCATCTGCCAT-3’) and the control MO exons (Fig. 1C and Table A2). Based on these data, we confirmed carrying the reverse sequence (5’-TACCGTCTACCTAAAAGATGA- that the splice junction sites of the vertebrate CFLAR genes are well GACC-3’) were obtained from GeneTools LLC (Philomath, OR) and conserved in both the second DED motif and a CASc* domain, al- dissolved at a concentration of 500 mM in 1 Danieau’s buffer though they vary in position within the non-functional hinge re-

[5 mM Hepes (pH 7.6), 58 mM NaCl, 0.7 mM KCl, 0.6 mM Ca(NO3)2, gion (Fig. 1C). Consequently, the organization of the CFLAR genes is and 0.4 mM MgSO4]. One nanoliter of this solution or evolutionarily conserved and maintained in the four living classes 1 Danieau’s buffer was injected into one- or two-cell embryos, of vertebrates, excluding unexamined reptiles. Taken together, our which were prepared as described [38], before allowing the em- results suggest that c-FLIP proteins retain a relatively unchanged bryos to develop at 23 °C. structure during vertebrate evolution.

2.13. Statistical analyses 3.2. Molecular phylogenetic analysis of c-FLIP with its paralogous proteins Data are presented as the means and standard deviations from at least three independent experiments. Significant differences In previous studies, we proposed that the CFLAR/c-FLIP, CASP8, between treatment groups were evaluated by calculating P values CASP10, and CASP18 genes diverged from the ancestral gene by based on Student’s t-test. gene duplication during evolution of early vertebrates, and the CARD-casp8 (tentatively named) gene in the fish lineage is also a diverged gene because it is located next to the casp8 gene, with 3. Results sequence similarity in the protease domain [16,18]. To better understand the evolutionary conservation of c-FLIP proteins in ver- 3.1. Isolation of the CFLAR/c-FLIP genes from non-mammalian vertebrates, we constructed a molecular phylogenetic tree composed tebrates and the assessment of similarities of the CASc*/CASc domains of c-FLIP and its paralogous proteins, CASP8, CASP10, CASP18, and CARD-casp8. For construction of the To investigate the distribution of genes orthologous to human tree, we included additional c-FLIP protein sequences from various CFLAR in animals, the NCBI DNA database was searched using primitive fishes, the lamprey (P. marinus, Cyclostomata), the cat- TBLASTN. Candidate EST clones in the chicken (G. gallus), African shark (S. canicula, Chondrichthyes), the spotted gar (L. oculatus, clawed frog (X. laevis), medaka (O. latipes), and stickleback (G. Actinopterygii), and the coelacanth (L. chalumnae, Sarcopterygii) aculeatus) were identified. Sequencing of these EST clones led to predicted from the genome and transcriptome databases, as listed the determination of the complete open reading frames encoding in Table A1. In addition to known amino acid sequences, we also 493, 458, 460, and 469 amino acids, respectively. Each of the searched for unreported homologs in a set of sequence databases predicted amino acid sequences exhibited the highest similarity to to identify new members in the caspase-8 superfamily (Table A1). 3 the long form of human c-FLIP in a reciprocal BLASTP analysis. As The assembled molecular phylogenetic tree clearly indicated that shown in Fig. 1A, the multiple alignment of amino acid sequences the c-FLIP subfamily forms a unique clade distinct from the other of these proteins with human and zebrafish c-FLIPs verified high subfamilies consisting of CASP8, CASP10, CASP18, and CARD-casp8 similarity in two DED motifs and an inactive CASc (CASc*) domain. (Fig. 2A), suggesting that the c-FLIP subfamily members are in- Therefore, these proteins are non-mammalian orthologs of c-FLIP. dependently evolving. In addition, the longer branch lengths of the To assess the structural similarity between non-mammalian c-FLIP subtree suggest that the amino acid changes in c-FLIP have c-FLIP proteins and human c-FLIP, three-dimensional structural quickly progressed compared with the other paralogous proteins models were constructed and compared with the crystal structure after the divergence of the c-FLIP clade from the other subfamilies of human c-FLIP (Fig. 1B). The models showed that the core (Fig. 2A and B). Furthermore, comparison of the species divergence structural elements of non-mammalian CASc* domains comprise between CASc* and CASc domains revealed that the molecular β fi α six -sheets and ve -helices. When these structural models evolutionary rate of c-FLIP is 1.7-fold higher than that of CASP8 were superimposed with a crystal structure of the human CASc* when were analyzed in human, mouse, and chicken (Table 1). domain, they showed a resemblance of the overall fold. Thus, the Thus, c-FLIP seems to have evolved in its own particular way prediction of the spatial structure indicates that the tertiary compared to the other paralogous molecules in vertebrates. structure of the c-FLIP family members is conserved from fish to mammals, and is consistent with the analysis of the primary amino acid sequence (see below). 3.3. The evolutionarily conserved amino acid residues of c-FLIP fa- In the CFLAR-paralogous CASP8 gene, new intron insertions mily proteins have occurred in the coding region during teleost evolution [26]. In addition, both Casp10 and CASP18 paralogous genes have been Both histidine (H) and cysteine (C) residues in the CASc of caspases form a catalytic dyad and are necessary for catalysis as a 3 Data not shown cysteine protease (Fig. 3A) [39]. These two amino acid residues K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189 179

Fig. 1. Analysis of the protein structures of c-FLIP proteins. (A) Multiple alignment of amino acid sequences of human, chicken, African clawed frog, medaka, stickleback, and zebrafish c-FLIP proteins. Identical and similar amino acids in all family members are indicated by red and blue, respectively. The two bold lines and a yellow box indicate the DED motif and the protease-like CASc* domain, respectively. The numbers (1) and (2) shown above the sequences indicate the crucial arginine and tyrosine amino acid residues, which are the cause of the deactivation of the protease activity [8], and an asterisk indicates the conserved leucine residue. (B) Structural superposition of human c-FLIP and non-mammalian c-FLIP proteins. Structural models of the CASc* domain of chicken (light pink), clawed frog (orange), and zebrafish (magenta) c-FLIP proteins were computationally generated and superimposed with the CASc* domain of human c-FLIP (cyan, PDB ID: 3H13). (C) Comparison of the exon-intron organization of the CFLAR genes. The splice junction sites of the CFLAR/c-FLIP genes in vertebrates are indicated by vertical lines. Their positions were defined by the comparison of the respective genomic and cDNA sequences from the species listed in Table A2. The regions corresponding to two DED motifs and a CASc* domain are indicated by blue and red boxes, respectively. 180 K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189

Fig. 2. Molecular phylogenetic analysis of c-FLIP and its paralogous proteins. (A) A molecular phylogenetic tree of c-FLIP and its paralogs, CASP8, CASP10, CASP18, and CARD- casp8 was constructed by RAxML, based on an alignment of the protease-like/protease (CASc*/CASc) domains (145 amino acid sites without mismatches including insertion/ deletion (indel)). Human CASP3 and amphioxus CASP3-like AmphiCASP3/7 proteins were used as an out-group for rooting the tree. Numbers (in orange) at internal branches indicate the percentage of support of that branching pattern based on a bootstrap method with 100 replicates (bootstrap probabilities). The scale bar indicates the evolutionary distance of 0.2 amino acid substitutions per site. (B) A molecular phylogenetic tree restricted to the c-FLIP subfamily proteins. The tree was constructed by RAxML, based on an alignment of the c-FLIP CASc* domains (164 amino acid sites) derived from 17 animal species including zebrafish. The number (in blue) above each branch shows branch length, while the number (in orange) beneath each internal branch shows bootstrap probability. The scale bar indicates the evolutionary distance of 0.2 amino acid substitutions per site. The identification numbers of the c-FLIP subfamily and its paralogous proteins shown in the tree are listed in Table A1. Abbreviations of the species: A. ca, A. carolinensis (anole lizard); A.mi, A. mississippiensis (alligator); B.fl, B. floridae (lancelet); D.re, D. rerio (zebrafish); G.ac, G. aculeatus (stickleback); G.ga, G. gallus (chicken); H.sa, H. sapiens (human); L.ch, L. chalumnae (coelacanth); L.er, L. erinacea (little skate); L.oc, L. oculatus (spotted gar); M.do, M. domestica (opossum); M.ga, M. gallopavo (turkey); M.mu, M. musculus (mouse); O.la, O. latipes (medaka); R.no, R. norvegicus (rat); S.ca, S. canicula (catshark); S.sc, S. scrofa (pig); X.la, X. laevis (African clawed frog) were conserved in the active site of all CASP8, CASP10, CASP18, Therefore, these two amino acids were likely conserved after and CARD-casp8 proteins.4 By comparing amino acid sequences, evolving in bony vertebrates including bony fish and tetrapods both arginine (R) and tyrosine (Y) residues (R315 and Y360 in hu- (Actinopterygii and Sarcopterygii). Furthermore, the substitution man c-FLIP), located in the inactive CASc* domain of c-FLIP pro- from arginine to leucine has uniquely occurred in the mouse be- teins, replace the histidine and cysteine residues in the other cause the rat, which is a closely allied species to the mouse, has subfamilies (Fig. 3A) [8], and are preserved in most animals ex- retained the arginine residue. The time of divergence between amined, except lamprey, catshark, and mouse (Fig. 3B and Fig. A1). these two species is estimated at 23–33 million years ago [40,41]. The lamprey and catshark, which belong to the Cyclostomata and As it was thought that the arginine and tyrosine residues are Chondrichthyes, respectively, exhibited no conservation. crucial for the function of c-FLIP, the structural arrangement sur- rounding these amino acid residues was examined by computa- 413 4 Data not shown tional analysis. For this analysis, the leucine residue (L in K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189 181

Table 1 3.5. Non-mammalian c-FLIP proteins function as NF-κB activators Comparison of evolutionary distances (Poisson correction) and molecular evolu- # tionary rates of protease-like/protease (CASc /CASc) domains. It was previously reported that human c-FLIP induces NF-κB

compared species CASP8 CASP10 c-FLIP activation [14,15]. To investigate whether non-mammalian c-FLIP proteins are also able to induce NF-κB activation, we examined human–mouse 0.239 (1.00) 0.253# (1.06) 0.434 (1.82) their potential in transfected cells with the dual-luciferase re- human–chicken 0.392 (1.00) 0.533 (1.36) 0.688 (1.76) porter assay system (Fig. 5A, B). Exogenous expression of either mouse–chicken 0.460 (1.00) 0.592# (1.29) 0.733 (1.59) chicken or clawed frog c-FLIP proteins increased the luciferase Average 1.24 1.72 activity in transfected HEK293 cells (Fig. 5A). This indicated that Evolutionary distances were calculated for highly homologous 179 amino acid sites NF-κB acquired transcriptional activity in the presence of the without mismatches including insertion/deletion (indel). Each value in parentheses exogenous c-FLIP proteins. By changing the transfection reagent, is relative molecular evolutionary rate normalized by the evolutionary distance of we re-examined whether zebrafish c-FLIP is able to induce NF-κB CASP8. # activation. In these experiments, c-FLIP proteins derived from For CASP10, the amino acid sequence data of the naked mole rat (Hetero- fi fi cephalus glaber) was used instead of the mouse because the Casp10 gene has been zebra sh and the other two sh, medaka and stickleback, in- lost in mice. creased the luciferase activity in transfected cells (Fig. 5B). To confirm NF-κB activation by non-mammalian c-FLIP proteins, the human) was also examined since it is conserved throughout ver- intracellular localization of p65 (RelA), an NF-κB subunit, was tebrates (Fig. A1). Modeling verified that these three residues, ar- checked in non-mammalian c-FLIP-expressing cells. We co-trans- ginine, tyrosine, and leucine, are capable of forming a “pseudo- fected a plasmid, pEGFP/p65 encoding a fusion protein, EGFP/p65, fl catalytic triad” (Fig. 3C). By contrast, mouse and lamprey c-FLIP consisting of enhanced green uorescent protein (EGFP) and p65, proteins may be unable to form such a firm structure (Fig. 3C). with plasmids encoding non-mammalian c-FLIP proteins into Thus, with the exception of mouse, c-FLIP proteins have evolu- HEK293 cells. The EGFP/p65 protein translocates to the nucleus κ tionarily conserved the spatial arrangement of amino acids re- when the NF- B signaling pathway is activated [35]. As shown in quired not only for the skeletal structure, but also for the triad Fig. 5C and D, EGFP/p65 nuclear localization increased in non- formation, while they nevertheless show characteristic differences mammalian c-FLIP-expressing cells relative to control cells. In this from the other subfamily members including CASP8, CASP10, assay system, human c-FLIP also stimulated the nuclear translo- CASP18, and CARD-casp8. cation of EGFP/p65. Furthermore, the interaction between tumor necrosis factor receptor-associated factor 2 (TRAF2) and non- mammalian c-FLIP proteins was examined. Previous studies have 3.4. Non-mammalian c-FLIP proteins play a role as negative reg- shown that TRAF2 plays a crucial role in NF-κB activation [46], and ulators of apoptosis human c-FLIP associates with TRAF2 [47]. We confirmed their interactions by coimmunoprecipitation and immunoblot analyses c-FLIP plays a role as a regulator of CASP8 activation mediated (Fig. 5E). TRAF2 proteins were detected in the precipitated com- through death receptors [42]. We examined the anti-apoptotic plex. Taken together, these results suggest that non-mammalian activity of non-mammalian c-FLIP proteins in human HeLa cells. c-FLIP proteins have the ability to induce NF-κB activation in hu- First, the ectopic expression of chicken, African clawed frog, and man cells. zebrafish c-FLIP proteins in transfected HeLa cell was confirmed by immunoblot analysis. To detect products in cultured cells, each 3.6. Involvement of c-FLIP in embryonic development c-FLIP was tagged with the Flag epitope (Fig. 4A). Proteins with the predicted size were detected (Fig. 4B). We then determined their In the mouse, c-FLIP is critical for embryonic development. ability to protect cells from death receptor-mediated apoptotic c-FLIP deficiency leads to embryonic lethality due to irregular induction by examining the viability of transfectants subjected to development of the heart [48]. To more comprehensively under- Fas-ligation. Control cells expressing only Venus, which is a variant stand the physiological roles of c-FLIP during embryonic devel- fl of yellow uorescent protein [43], were sensitive to Fas-induced opment in vertebrates, we investigated the expression profiles of apoptosis, resulting in no Venus-positive cells after apoptotic sti- CFLAR/c-FLIP transcripts during embryogenesis of non-mammals. fi muli (Fig. 4C). In contrast, chicken, frog, or zebra sh c-FLIP-ex- First we isolated total RNAs from embryos of African clawed frog pressing HeLa cells survived even in the presence of an agonistic (X. laevis) at various stages of development, and examined CFLAR anti-Fas antibody, as evidenced by the detection of only Venus- mRNA by RT-PCR. In X. laevis embryos, CFLAR mRNA was detected positive cells, while untransfected cells underwent apoptosis in in all samples prepared from stages 0, 3, 9, 13, 18, 25, and 30 the same culture dish. Similar results were observed in cells ex- (Fig. 6A). These data indicated that CFLAR mRNA exists in both pressing medaka or stickleback c-FLIP (Fig. A2). These data in- maternal and zygotic transcripts during embryogenesis. To de- dicated that non-mammalian c-FLIP proteins as well as human termine if CFLAR/c-FLIP is critical in embryogenesis, we generated c-FLIP are able to protect mammalian cells from Fas-mediated a plasmid construct encoding a truncated form of c-FLIP, FLIP apoptotic induction. FADD is a pro-apoptotic adapter molecule (DEDs), which contains only the two DED motifs (Fig. 6B), and responsible for recruiting CASP8, CASP10, or c-FLIP into the death synthesized mRNA using this plasmid as a template. The resulting receptor complex through homotypic DED–DED interactions truncated mutant protein, lacking the CASc* domain, was expected [44,45] in response to extrinsic apoptotic stimuli. We investigated to dysregulate the function of full-length endogenous c-FLIP in the interaction between non-mammalian c-FLIP proteins and hu- organisms. In previous studies, discordant results have been re- man FADD by coimmunoprecipitation and immunoblot analyses. ported, with one concluding that the amino-terminal half of hu- Human FADD was detected in the c-FLIP complex precipitated man c-FLIP is a weaker NF-κB activator than full-length c-FLIP [14], with an anti-Flag antibody (Fig. 4D), suggesting that non-mam- whereas another study concluded that a truncated form con- malian c-FLIP proteins as well as human c-FLIP are able to interact tributed to higher NF-κB activation than full-length [49]. When an with FADD. Taken together, these results suggest that c-FLIP pro- amphibian truncated form of c-FLIP was examined for its ability to teins derived from birds, amphibians, and fish functionally act as induce NF-κB activation, this mutant induced NF-κB activation to anti-apoptotic molecules in the extrinsic apoptotic pathway by the same extent as full-length c-FLIP in mammalian HEK293 cul- interacting with pro-apoptotic molecules. ture cells (Fig. 6C). Following microinjection of mRNAs for FLIP 182 K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189 K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189 183

Fig. 4. Assessment of the anti-apoptotic activity of the c-FLIP subfamily proteins. (A) The protein structure of Flag-tagged non-mammalian c-FLIP proteins consisting of two DED motifs and a CASc* protease-like domain. (B) Immunoblot analysis of non-mammalian c-FLIP proteins. The empty vector, pCMV-Flag/HsFLIP, pME18S-Flag/GgFLIP, pME18S-Flag/XlFLIP, or pME18S-Flag/DrFLIP were transiently transfected into HeLa cells. After culturing for 48 h, transgene products were analyzed by immunoblotting with an anti-Flag antibody. (C) Cytological analysis of transfectants expressing non-mammalian c-FLIP proteins. HeLa cells expressing Venus with or without human, chicken, clawed frog, and zebrafish c-FLIP were incubated in the presence (lower panels) or absence (upper panels) of anti-Fas antibody and CHX for 8 h, and examined for viability by monitoring Venus-positive cells. Both phase-contrast and fluorescence images in the same field were captured under the microscope. Scale bars represent 100 μm. (D) Co- immunoprecipitation and immunoblot analysis of physical interactions between non-mammalian c-FLIP proteins and human FADD. Human HEK293 cells were co-transfected with pME18S-HA/hFADD in conjunction with pME18S empty vector, pCMV-Flag/HsFLIP, pME18S-Flag/GgFLIP, pME18S-Flag/XlFLIP, or pME-Flag/DrFLIP. Baculovirus p35 was introduced into all transfected cells to prevent cell death. After 2 days of cultivation, transfected cells were harvested and lysed in lysis buffer. The cell lysates were immunoprecipitated with an anti-FLAG M2 affinity gel. Coimmunoprecipitates and aliquots of cell lysates were examined by immunoblot analysis with anti-Flag, anti-HA, and anti-actin antibodies, respectively. An asterisk indicates immunoglobulin light-chain. Abbreviations: H, human; C, chicken; F, clawed frog; Z, zebrafish; IP, immunoprecipitation; WB, western immunoblotting.

(DEDs) into the four-cell-stage blastocysts, the morphology of Thus, our results suggest that CFLAR/c-FLIP is involved in the early embryos was examined after developing to stage 45. Injection into development of clawed frog embryos. the equatorial areas of the two dorsal blastomeres caused abnor- We also investigated the function of the fish c-Flip protein in mal development of embryos associated with thorax edema embryonic development. The expression profile of cflar/c-Flip (Fig. 6D, middle panel). In contrast, abnormal embryos associated transcripts during embryogenesis in zebrafish was examined with scoop of the abdomen were observed after microinjection of (Fig. 7A). During development, cflar transcripts were detected in the mRNA into the ventral area (Fig. 6D, lower panel). These all samples prepared from the cleavage, gastrula, segmentation, phenotypes appeared in a dose-dependent manner (Fig. 6E). These pharyngula, and hatching periods. These data indicate that cflar data clearly indicate that irregular development of embryos occurs mRNA exists in both maternal and zygotic transcripts during em- frequently following overexpression of a truncated form of c-FLIP. bryogenesis of fish. To understand the physiological role of cflar/c-

Fig. 3. The specified amino acid substitutions of the CAScn domain and their evolutionary conservation. (A) A schematic diagram of human CASP8 and c-FLIP proteins. Two DED motifs, a CASc protease domain, and a CASc* protease-like domain are indicated by boxes, respectively. White stars indicate the position of the essential amino acid residues, histidine (H) and cysteine (C) for the catalytic dyad formation in CASP8, whereas the gray stars indicate arginine (R) and tyrosine (Y) of c-FLIP positionally corresponding to two amino acid residues of CASP8, and are also coincident with positions (1) and (2), as shown in Fig. 1A. (B) A summary of critical amino acid residues of c-FLIP proteins conserved in vertebrates. Both the arginine (R) and tyrosine (Y) residues are evolutionarily conserved in most bony vertebrates. In the mouse c-Flip protein, the arginine residue is exceptionally changed to leucine. In the fish lineage, a phenylalanine (F) residue instead of tyrosine is present in the catshark whereas both lysine (K) and valine (V) residues are present at these two critical positions in the lamprey. A taxonomic tree of the species shown at the left was generated based on the previous study [70]. (C) Closeup views of the pseudocatalytic triad of c-FLIP proteins. The arginine, tyrosine, and leucine residues, which are shown in spheres, in human, chicken, clawed frog, and zebrafish c-FLIP proteins are brought close together in the three-dimensional shape forming a triad. In the lamprey, c-FLIP includes lysine, valine, and cysteine residues instead of the conserved amino acids, resulting in the failure of the triad formation. As mouse c-FLIP has replaced the arginine residue with leucine (L), there is little interaction with another leucine. The gray, red, and blue spheres indicate C, O, and N atoms, respectively. 184 K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189

Fig. 5. Functional analyses of non-mammalian c-FLIP proteins on NF-κB activation ability. (A, B) Enzymatic analysis of NF-κB activation induced by non-mammalian c-FLIP proteins. The empty vector or plasmids carrying c-FLIP were transiently co-transfected with pNFκB-Luc and pRL-TK into HEK293 cells, and cultured for 48 h. NF-κB activation was analyzed by measuring enzyme activities of dual luciferases produced in transfected cells using a luminometer. Data are presented as the means and standard deviations of samples counted from three independent experiments. The statistically-significant difference between two groups was evaluated by Student’s t-test. (C) Cytological analysis of a NF-κB component, p65, in cells expressing c-FLIP. HEK293 cells were transiently transfected with an empty vector or plasmids carrying c-FLIP together with pEGFP/p65, and cultured for 24 h. The localization of EGFP/p65 proteins in transfected cells was analyzed by fluorescence microscopy. Typical patterns of subcellular localization of EGFP/p65: EGFP/p65 normally localizes in the cytoplasm (upper panels), but it translocates into the nucleus when the NF-κB signaling pathway undergoes activation (lower panels) [35]. Scale bars indicate 20 μm. (D) A summary of cytological analyses on the translocation of EGFP/p65. Within each field, positive cells (dark gray) and negative cells (light gray) for EGFP/p65 translocation were counted under the fluorescent microscope and percentages of total were calculated. N indicates the total number of transfected cells examined in four independent experiments. (E) Co-immunoprecipitation and immunoblot analysis of physical interactions between non- mammalian c-FLIP proteins and mouse TRAF2. HEK293 cells were transfected with either pCMV-Flag/HsFLIP, pME18S-Flag/GgFLIP, pME18S-Flag/XlFLIP, pME18S-Flag/DrFLIP, or control vector in combination with pME18S-Myc/TRAF2. Forty-eight hours after transfection, cells were lysed and the c-FLIP complex was immunoprecipitated from whole cell lysates with an anti-Flag antibody. Samples were analyzed alongside aliquots of the cell lysates by immunoblotting with anti-Flag, anti-Myc, and anti-actin antibodies, respectively. An asterisk indicates immunoglobulin light-chain. Abbreviations: H, human; C, chicken; F, clawed frog; Z, zebrafish; IP, immunoprecipitation; WB, western immunoblotting. K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189 185

Fig. 6. Irregular development of X. laevis embryos expressing a c-FLIP mutant. (A) The expression profile of African clawed frog CFLAR transcripts during embryogenesis. Total RNAs isolated from X. laevis embryos, which were collected at indicated stages (lanes 2–8), was analyzed by RT-PCR. PCR products amplified with primers specific for CFLAR (upper panel) and ODC (lower panel) were resolved by acrylamide-gel electrophoresis. Molecular weight markers were run in lane 1, and a negative control (NC) with no polymerase was run in lane 9. Arrows indicate the expected molecular weights of the CFLAR and ODC PCR products, respectively. (B) The structure of a truncated c-FLIP mutant, FLIP(DEDs), consisting of only two DED motifs. (C) Effect of a FLIP(DEDs) mutant on NF-κB activation. The empty vector or plasmids encoding intact or truncated forms of clawed frog c-FLIP were transiently co-transfected with pNFκB-Luc and pRL-TK into HEK293 cells. After 48 h in cell culture, NF-κB activation was analyzed by measuring enzyme activities of dual luciferases produced in transfected cells using a luminometer. Data are presented as the means and standard deviations of samples counted from three independent experiments. The statistically-significant difference between two groups was evaluated by Student’s t-test. (D) Morphological analysis of embryos expressing FLIP(DEDs). X. laevis embryos were injected without (upper panel) or with mRNA encoding FLIP(DEDs), at the equatorial area of two dorsal (middle panel) or two ventral (lower panel) blastomeres at the four-cell-stage. Images of the developing embryos were acquired at stage 45. Arrowheads indicate the edema and abdominal constriction of the injected embryos, respectively. Scale bars indicate 1 mm. (E) A summary of the phenotypic data presented in (D). Embryos displaying edema (red), abdominal constriction (dark blue), bending (light blue), and other defects (black) were counted under the microscope. Data represent the percentages calculated from five independent experiments. Abbreviations: D, dorsal; V, ventral.

Flip in fish embryos, we examined the effects of an antisense (Fig. 7C). Thus, the deregulation of c-FLIP expression derailed morpholino oligonucleotide specifictocflar/c-Flip, cflar-MO, in the embryonic development, suggesting the involvement of c-FLIP in developing embryos. We injected cflar-MO into the fertilized eggs embryogenesis. and examined the morphology of embryos during development. We observed abnormal development of the injected embryos in a 4. Discussion dose-dependent manner, resulting in thorax edema and the de- layed velocity of red blood cell flow or the clogging of red blood In this study, in order to understand the ubiquity and diversity cells (Fig. 7B). We statistically confirmed these abnormalities of the c-FLIP/CFLAR subfamily members during vertebrate 186 K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189

Fig. 7. Developmental anomalies in zebrafish embryos with knockdown of cflar transcripts. (A) The expression profile of zebrafish cflar transcripts during embryogenesis. Total RNAs isolated from embryos, which were collected at 1, 6, 18, 36, and 48 h after fertilization (lanes 2–6), was analyzed by RT-PCR. PCR products amplified with primers specific for cflar (upper panel) and tubulin α6 (tubα6) (lower panel) were resolved by agarose-gel electrophoresis. Molecular weight markers were run in lane 1, and a negative control (NC) with no template DNA was run in lane 7. Arrows indicate the expected positions of the cflar and tubα6 PCR products, respectively. (B) Morphological analysis of developing embryos subjected to morpholino oligonucleotide (MO) injection. Fertilized eggs were injected with an antisense MO for cflar, cflar-MO and their development was monitored under the microscope. Images of the developing embryos were acquired at three days later. The resulting abnormal phenotypes consisted of edema (middle panel) and a cluster of blood cells in the vessel (right panel), indicated by an arrow. (C) A summary of the phenotypic data presented in (B). Embryos displaying the edema (red) or irregular flow of red blood cells (blue) were counted under the microscope. Data represent the percentages calculated from four independent experiments. evolution, we identified and characterized non-mammalian c-FLIP the other hand, the phylogenetic analysis verified that, compared proteins. The three-dimensional structure analysis showed a si- with paralogous proteins such as CASP8, c-FLIP proteins have a milar skeletal structure in vertebrate c-FLIP proteins. In addition, faster substitution rate of amino acids in the CASc*/CASc domains, the arginine and tyrosine residues located in the pseudocatalytic although they have preserved several amino acids that might be triad of the CASc* domain are conserved in most bony vertebrates. critical for maintenance of the structure and functionality. Con- Non-mammalian c-FLIP proteins functionally retain their potential sequently, c-FLIP, which arose at an early stage of vertebrate as both apoptosis-regulating molecules and inducers of NF-κB evolution, still might be continuing its molecular evolution while activation. These results provide evidence that c-FLIP proteins are keeping the core structures. evolutionarily conserved in protein structure and function across In our previous studies, we proposed that the CFLAR, CASP8, vertebrate species. This prompts the hypothesis that these con- CASP10, and CASP18 genes have been tandemly-duplicated from a served functions were already established in the fish ancestor. On single ancestral gene in the course of vertebrate evolution [16], K. Sakamaki et al. / Biochemistry and Biophysics Reports 3 (2015) 175–189 187 and we and another group showed that both CFLAR and CASP8 5. Conclusions genes invariably exist in vertebrates, whereas the other two genes, CASP10 and CASP18 have been lost in some mammals [16–18]. The We demonstrated the universal functionality of c-FLIP proteins main function of c-FLIP seems to be the regulation of CASP8 ac- during vertebrate evolution. In vertebrates, the c-FLIP subfamily tivation, but it is restricted to the vertebrate lineage. As supporting members commonly function as a negative regulator of the ex- evidence for such a lineage specificity, no orthologs of c-FLIP have trinsic apoptosis associated with CASP8 activation, and as an in- κ been reported in the fruit fly Drosophila melanogaster and ascidian ducer for NF- B activation. Although these physiological actions of fi Ciona intestinalis databases, while CASP8 exists in both animals c-FLIP proteins might be the result of the de ciency of protease fi [50, 51]. To understand the appearance and the acquisition of the activity, the speci c amino acid substitutions in the catalytic dyad functional properties of c-FLIP in the process of evolution, the of the caspase domain and their conservation during evolution investigation of jawless fish, such as lamprey and hagfish, is under selective pressure are striking, suggesting the possibility that c-FLIP has acquired a new function, not merely the loss of the helpful because lamprey c-FLIP protein has not yet been char- enzyme activity, as was proposed previously [68]. Previous studies acterized with regard to its functionality, and it clearly exhibits have shown that regardless of cell death, c-FLIP functions as a differences in the protein structure relative to other c-FLIP modulator of the Wnt signaling pathway [69], and an activator of proteins. the Erk signaling pathway [15]. Therefore, future studies should be c-FLIP was previously identified as a regulator of death re- directed at investigating the molecular mechanisms by which ceptor-mediated apoptosis. Whereas the deficiency of these death c-FLIP acts as a new functional molecule with interactions apart receptors causes no irregular embryogenesis [52], c-FLIP-deficient from caspase signaling. mice died at mid-gestation during embryogenesis due to irregular development of the heart [48]. This suggests that c-FLIP functions during embryonic development independent of death receptor- Acknowledgments mediated signal transduction. A recent study reported that c-FLIP is indispensable for limited activation of CASP8 during embryonic The authors are grateful to N. Inohara (University of Michigan), development of rodents [53]. Therefore, c-FLIP and CASP8 act in a J. Inoue (University of Tokyo), A. Miyawaki (RIKEN), J.A. Schmid coordinated manner during embryogenesis. Apart from their re- (Medical University of Vienna), and K. Nomura (Kyushu University) spective roles in apoptosis, these proteins suppress necroptotic cell for providing the zebrafish cflar, TRAF2, and Venus cDNAs, a EGFP/ death mediated by receptor-interacting serine/threonine kinases p65 reporter plasmid, and a FAD-II monoclonal antibody, and Y. 1 and 3 (RIPK1 and RIPK3) are involved in [53–55]. Necroptosis is a Satou, M. Furutani-Seiki, A. Momoi, H. Yamashita, and S. Sakata for form of regulated or programmed necrosis and its signaling their technical assistance. We also thank to J.A. Hejna (Kyoto pathway has been defined [55–61]. When CASP8 is inactive and University) for critical reading of the manuscript. The complete hence unable to control RIPK1, the necroptotic signaling pathway sequence of the stickleback caspase-10 cDNA (GenBank Accession is activated: RIPK1 is free to interact with and phosphorylate Number: HQ285318) has been deposited in the GenBank database. RIPK3, and activated RIPK3 phosphorylates and activates an ef- This work was supported in part by Platform for Drug Discovery, fector molecule, mixed lineage kinase domain-like (MLKL), re- Informatics, and Structural Life Science from the Ministry of Edu- sulting in cell death. These core components required for ne- cation, Culture, Sports, Science and Technology, Japan. croptosis are also detectable in non-mammalian vertebrates by searching the database.5 It is thus likely that a major developmental role of vertebrate c-FLIP in conjunction with CASP8 may be Appendix A. Supplementary material to jointly antagonize the induction of necroptotic cell death, although the molecular mechanism of the c-FLIP and CASP8 co- Supplementary data associated with this article can be found in ordination still remains elusive [55, 60–62]. Interestingly, we no- the online version at doi:10.1016/j.bbrep.2015.08.005. ticed that the phenotypes of clawed frog embryos expressing a truncated mutant of c-FLIP are morphologically similar to those of embryos exogenously expressing a protease-deficient mutant of References CASP10β [63]. This observation suggests that a CASP8 paralog, CASP10 also works with c-FLIP in developing embryos. Thus, c-FLIP [1] N.A. Thornberry, Y. Lazebnik, Caspases: enemies within, Science 281 (1998) 1312–1316. might have pivotal roles not only for regulation of apoptosis as- [2] R.C. Taylor, S.P. Cullen, S.J. Martin, Apoptosis: controlled demolition at the sociated with CASP8 activation but also for development and/or cellular level, Nat. Rev. Mol. Cell Biol. 9 (2008) 231–241. proliferation. [3] L. Galluzzi, I. Vitale, J.M. Abrams, E.S. Alnemri, E.H. Baehrecke, M. V. Blagosklonny, T.M. Dawson, V.L. Dawson, W.S. El-Deiry, S. Fulda, E. 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